Jun Jong Hwa, Sohn Wern-Joo, Lee Youngkyun, Chang Sung Dong, Kim Jae-Young
Department of Ophthalmology, School of Medicine, Dongsan Medical Center, Keimyung University, Daegu, Republic of Korea.
Cell Tissue Res. 2014 Jul;357(1):101-8. doi: 10.1007/s00441-014-1870-4. Epub 2014 May 6.
An in vitro culture model enabling posterior capsule opacification (PCO) to be investigated was developed and established by using low-melting-point (LMP)-agarose gel to support the capsular bag. After removal of the cornea from rodent and porcine eyeballs, the lens zonules were dissected. Whole lens explants were embedded into 2 % (37 °C) LMP-agarose gel solution. As performed routinely in cataract surgery, capsulotomy and lens fiber removal were carried out in the solidified LMP-agarose gel as sham cataract surgery. The LMP-agarose-gel-supported capsular bag/lens epithelial cell (CB-LEC) complexes were maintained in Dulbecco's modified Eagle medium supplemented with 10 % fetal bovine serum in an anterior face-down position. The proliferation and migration of LECs into the posterior capsule were observed every 12 h by phase-contrast microscopy. Epithelial cells were observed at the central portion of the CB-LEC complexes after 56.57 ± 16.56 h (n = 7) and 106 ± 14.03 h (n = 6) of culture, for rodent and porcine lenses, respectively. The solidified gel allowed clear microscopic observations and whole-mount immunostaining evaluations of the whole area of the capsular bag. Histological examinations revealed the proliferation, migration, and transdifferentiation of LECs related to posterior capsule opacification. This new in vitro culture model provides experimental benefits by maintaining the natural contour of the capsule without implants inside or outside of the capsule. In addition, this model system allows pharmacological and histological evaluations of the cultured CB-LEC complexes without additional manipulations.
通过使用低熔点(LMP)-琼脂糖凝胶来支撑囊袋,开发并建立了一种能够研究后囊膜混浊(PCO)的体外培养模型。从啮齿动物和猪的眼球上摘除角膜后,解剖晶状体悬韧带。将整个晶状体植入物嵌入2%(37℃)的LMP-琼脂糖凝胶溶液中。如同白内障手术常规操作那样,在固化的LMP-琼脂糖凝胶中进行囊切开术和晶状体纤维摘除,作为假白内障手术。将LMP-琼脂糖凝胶支撑的囊袋/晶状体上皮细胞(CB-LEC)复合物保持在前部朝下的位置,置于补充有10%胎牛血清的杜氏改良 Eagle培养基中。通过相差显微镜每12小时观察一次LEC向后囊膜的增殖和迁移情况。对于啮齿动物和猪的晶状体,分别在培养56.57±16.56小时(n=7)和106±14.03小时(n=6)后,在CB-LEC复合物的中央部分观察到上皮细胞。固化的凝胶允许对囊袋的整个区域进行清晰的显微镜观察和全层免疫染色评估。组织学检查揭示了与后囊膜混浊相关的LEC的增殖、迁移和转分化。这种新的体外培养模型通过保持囊膜的自然轮廓,且在囊膜内外均无植入物,提供了实验优势。此外,该模型系统允许对培养的CB-LEC复合物进行药理学和组织学评估,而无需额外操作。